ABSTRACT
A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB-DEG, PHB-TEG, PHB-PEG, and PHB-HV-PEG copolymers using short-chain PEGs (with n ≤ 9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB-HV-PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.
Subject(s)
Azotobacter/metabolism , Hydroxybutyrates/metabolism , Polyesters/metabolism , Chromatography, Gel , Hydroxybutyrates/chemistry , Molecular Weight , Polyesters/chemistry , Proton Magnetic Resonance SpectroscopyABSTRACT
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.
ABSTRACT
A biodegradable polymer of bacterial origin, poly(3-hydroxybutyrate) (PHB), is intensively studied as biomaterial for tissue engineering. However, factors determining its biocompatibility still require better understanding. To analyze the PHB films biocompatibility, the polymer material was modified by hydrophilic polymer, poly(ethylene glycol) 300 (PEG). The blends PHB/PEG with different PEG content (10, 20, 30 and 50%) were produced by subsequent incubation in water resulted in removal of 95% PEG. The surface roughness and hydrophilicity were studied by atomic force microscopy (AFM) and contact angle "water-polymer" measurement, respectively. The film biocompatibility on cell culture of COS-1 fibroblasts was studied in vitro. It was shown that both roughness and hydrophobicity are directly proportional to initial PEG content in the PHB/PEG blends. The growth rate of COS-1 fibroblasts on polymer films is determined by combination of two basic physicochemical properties of the polymer surface: the roughness and hydrophilicity. The optimal roughness requred for COS-1 cells growth is the average roughness more than 25 nm, whereas the limit values of the contact angle "water-polymer" that was responsible for relatively high cell viability were not found. These data indicate that the film surface roughness had the greatest effect on the cell growth, whereas the increase in the polymer surface hydrophilicity caused the additional positive effect on viability of attached cells. Thus, the modification of PHB polymer material by PEG resulted in the improved viability of cells cultivated on the polymer films in vitro. The obtained data can be used for development of such medical devices as surgeon patches and periodontal membranes.