Polihidroksialkanoatların Genetiği Değiştirilmiş Bakterilerde Uretimi, Karakterizasyonu ve Matematiksel Modellenmesi

Abstract

Owing to their poor degradation rates and their carcinogenic components, fossil-resource based plastics pose serious health problems on humans and the environment. Along with the depletion of fossil fuel reserves, polyhydroxyalkanoates (PHAs) are one of the eco-friendly alternatives to conventional plastics, because of their advantages such as biodegradability and biocompatibility. Polyhydroxyalkanoates (PHAs) are polyesters accumulated in a wide variety of microorganisms as intracellular carbon and energy storage material for the cells, under nutrient-limited conditions such as phosphorus, nitrogen and oxygen, and excess carbon source in the growth medium. The present study aims to produce PHA biopolymers from Bacillus megaterium NRRL B-14308 strain with a higher production efficiency, by investigating the bioprocess design parameters. Also, phaC gene was cloned into an expression vector, aiming to increase the production of PHA-synthase enzyme (PhaC) and thereby, increase the PHA production. Additionally, optimization of PHA production, metabolic modeling, purification and characterization studies were investigated using the recombinant strains. Structural and thermal properties of PHA purified by solvent extraction were characterized by FTIR, 1H-NMR, GC-FID, GC-MS, TGA and DSC analyses. The PHA characterization studies revealed that the PHA biopolymers were in PHB-co-PHV copolymer structure, consisted of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units. Fed-batch cultivations were performed in controlled bioreactors at pre-determined specific growth rates (µ=0.05, µ=0.075 and µ=0.1 h-1) to achieve a higher PHA yields. Accordingly, rec-B. megaterium cells in fed-batch fermentation with a pre-determined growth rate µ=0.1 h−1 produced the highest CDW (7.7 g L−1) and PHA concentration (6.15 g L−1). Moreover, an exponential glucose feeding profile resulted in 2.2-fold increase in PHA yield compared to batch cultivation. Also, the reconstructed genomic scale metabolic model for B. megaterium named iBM1128, with 7 genes, 6 metabolites, and 7 reactions added to the current iJA1121 model, was solved using metabolic flux balance analysis (FBA) technique. As a result, this study is signifcant for the production of PHB-co-PHV copolymer with a high 3HV content (58 mol% 3HV) by Bacillus strains from an unrelated, simple carbon source, glucose, with a defined medium and no need for precursor addition. Overall, this study paves the way to an enhanced biopolymer production process in B. megaterium cells, where the highest product yield on cell was obtained as YP/X = 0.74 g g−1.

Keywords: Biopolymer, polyhydroxyalkanoate, recombinant B. megaterium, bioprocess, characterization, metabolic modeling