Biosynthesis of poly(3-hydroxybutyrateco-3-hydroxy-4-methylvalerate) by Strain Azotobacter chroococcum 7B

Abstract

Production of novel polyhydroxyalkanoates (PHAs), biodegradable polymers for biomedical applications, and biomaterials based on them is a promising trend in modern bioengineering. We studied the ability of an effective strain-producer Azotobacter chroococcum 7B to synthesize not only poly(3-hydroxybutyrate) homopolymer (PHB) and its main copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), but also a novel copolymer, poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB4MV). For the biosynthesis of PHB copolymers, we used carboxylic acids as additional carbon sources and monomer precursors in the chain of synthesized copolymers. The main parameters of these polymers biosynthesis were determined: strain-producer biomass yield, polymer yield, molecular weight and monomer composition of the synthesized polymers, as well as the morphology of A. chroococcum 7B bacterial cells. The physico-chemical properties of the polymers were studied using nuclear magnetic resonance spectroscopy (NMR), differential scanning calorimetry (DSC), contact angle test, and other methods. In vitro biocompatibility of the obtained polymers was investigated using stromal cells isolated from the bone marrow of rats with the XTT cell viability test. The synthesis of the novel copolymer PHB4MV and its chemical composition were demonstrated by NMR spectroscopy: the addition of 4-methylvaleric acid to the culture medium resulted in incorporation of 3-hydroxy-4-methylvalerate (3H4MV) monomers into the PHB polymer chain (0.6 mol%). Despite the low molar content of 3H4MV in the obtained copolymer, its physico-chemical properties were significantly different from those of the PHB homopolymer: it has lower crystallinity and a higher contact angle, i.e. the physico-chemical properties of the PHB4MV copolymer containing only 0.6 mol% of 3H4MV corresponded to a PHBV copolymer with a molar content ranging from 2.5% to 7.8%. In vitro biocompatibility of the obtained PHB4MV copolymer, measured in the XTT test, was not statistically different from the cell growth of PHB and PHBV polymers, which make its use possible in biomedical research and development.